Physiological tissues and organs have adopted a variety of mechanisms for healing wounds and defects. For example, a cut in the skin may be healed by the formation of fibrous tissue, or scar, that connects the edges of the wound and protects interior tissues from the environment. Scar does not have the same mechanical or biological properties as undamaged skin. This healing mechanism, called repair, does not replace the structure or function of the original, undamaged tissue.
In contrast, organs such as liver have the ability to regenerate, replacing the wounded tissue with new, fully functional tissue having the same biological and mechanical properties as the original. Many tissues that typically heal by repair also have the ability to heal by regeneration. For example, small wounds such as paper cuts in the skin typically heal by regeneration, while larger wounds such as burns heal by formation of scar tissue. Furthermore, there are some tissues that do not properly heal at all on their own. To remedy defects and injuries in such tissue, physicians frequently attempt to artificially promote, repair or regeneration in such tissues.
In one example, articular cartilage lesions do not heal properly if left untreated, yet effective treatment methods remain an unsolved problem (Buckwalter, (1998) “Articular cartilage repair and transplantation.” Arthritis. Rheum. 41: 1331–1342). Tissue engineering (Langer, et al. (1993) “Tissue Engineering.” Science 260: 920–6) and gene therapy (Mulligan, (1993) “The basic science of gene therapy.” Science 260: 926–932) are two novel approaches to regenerate articular cartilage (see Evans, et al. (1999), “Genetically augmented tissue engineering of the musculoskeletal system” Clin. Orthop. 367 Suppl.:S410–418). Three-dimensional, functional cartilaginous tissue can be generated in bioreactors using isolated chondrocytes cultured on biodegradable scaffolds (Freed, (1997) “Tissue engineering of cartilage in space.” Proc. Natl. Acad. Sci. U S A 94: 13885–90; Vunjak-Novakovic, et al, (1999) “Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue engineered cartilage.” J Orthop. Res. 17: 130–138). The functional properties of the engineered constructs develop over the time of cultivation and come into the range of values measured for native articular cartilage only after several months of culture (Freed, 1997). Moreover, adult articular chondrocytes are problematic as a cell source for a clinical scenario of therapeutic tissue engineering due to their very low mitotic activity. However, isolated articular chondrocytes can be genetically modified (Baragi, et al. (1995) “Transplantation of transduced chondrocytes protects articular cartilage from interleukin 1-induced extracellular matrix degradation.” J. Clin. Invest. 96: 2454–60; Kang et al., (1997) “Ex vivo gene transfer to chondrocytes in full-thickness articular cartilage defects: A feasibility study.” Osteoarthritis Cartilage 5: 139–43; Doherty et al., (1998) “Resurfacing of articular cartilage explants with genetically-modified human chondrocytes in vitro.” Osteoarthritis Cartilage 6: 153–9; Madry, et al. (2000) “Efficient lipid-mediated gene transfer to articular chondrocytes.” Gene Ther. 7: 286–91) to express potentially beneficial genes (Doherty, 1998; Madry, 2000; Smith, et al. (2000) “Genetic enhancement of matrix synthesis by articular chondrocytes.” J. Rheumatol. 43: 1156–64). Insulin-like growth factor-I (IGF-I), a 7.6 kDa polypeptide growth factor, is a candidate gene to improve tissue engineering of cartilage, as it stimulates chondrocyte mitotic activity, increases proteoglycan and type-II collagen synthesis in vitro (Trippel, et al. (1997) “Growth factors as therapeutic agents.” Instr. Course. Lect. 46: 473–6) and enhances articular cartilage repair in vivo (Nixon, et al. (1999) “Enhanced repair of extensive articular defects by insulin-like growth factor-I-laden fibrin composites.” J. Orthop. Res. 17: 475–87). The application of chondrocytes that were genetically modified ex vivo to articular cartilage defects (Kang, 1997) is complicated due to technical challenges, such as the problem of cell loss after transplantation (O'Driscoll, et al (1998), “The healing and regeneration of articular cartilage.” J. Bone Joint Surg. Am. 80: 1795–12). Moreover, direct gene transfer into cartilage is difficult due to the dense matrix in which the chondrocytes are embedded (Ikeda, et al. (1998). “Adenovirus mediated gene delivery to the joints of guinea pigs.” J. Rheumatol. 25: 1666–73).
Gene transfer has been successfully applied to the tissue engineering of bioartificial muscle expressing a therapeutic protein (Powell, et al. (1999) “Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy.” Hum. Gene Ther. 10: 565–77) or to achieve the sustained release of plasmid DNA when associated with a biodegradable carrier in vivo (Bonadio, et al. (1999) “Localized, direct plasmid gene delivery in vivo: prolonged therapy results in reproducible tissue regeneration.” Nat. Med. 5: 753–9). Recent studies have demonstrated that isolated articular chondrocytes can be genetically modified (Baragi, 1995; Kang, 1997; Doherty, 1998; Madry, 2000) to express potentially beneficial genes (Baragi, 1995; Smith, 2000). Insulin-like growth factor-I (IGF-I), a 7.6 kDa polypeptide growth factor, stimulates chondrocyte mitotic activity, increases proteoglycan and type-II collagen synthesis in vitro (Trippel, 1997) and enhances articular cartilage repair in vivo (Nixon, 1999).
One approach to tissue engineering is to promote regeneration of lost or damaged tissue by providing materials that facilitate regenerative processes. For example, cells may be implanted into a wound site and allowed to generate extracellular matrix and other molecules that ordinarily form part of the normal tissue. Cells may also be taken from a patient or other source and utilized to synthesize tissue in vitro, following which the naturally synthesized tissue is transferred to the wound site.
Alternatively, a wound site may be implanted with a synthetic extracellular matrix that initially promotes migration of cells from the edges of a wound and later promotes normal metabolic and synthetic activity in the cells, enabling them to produce new tissue that will eventually replace the implanted matrix. Alternatively, these matrices may be coated with growth factors or other regulators designed to up-regulate certain metabolic activities in cells. They may also be coated with genetic material, which is then taken up into the cells where they control the production of desired proteins. In vitro cell cultures are also frequently transfected with DNA for specific growth factors or regulators. The DNA may be transferred directly into the cells or placed in the cell culture, where the cells uptake the genetic material through their membranes. However, the efficiency of cellular uptake of DNA varies, and some cells, such as chondrocytes, have not been demonstrated to uptake DNA immobilized on a cellular scaffold. Thus, it is desirable to employ more reliable transfection techniques for cells that are seeded in matrices for tissue engineered implants. It is hypothesized that the utilization of genetically engineered cells will improve the functionality of engineered tissue, while the incorporation of genetically engineered cells will improve cell delivery to the implantation site.